The metabolite patterns showed minor responses to OA in both life-cycle stages. Whereas previous gene expression analyses suggested that the observed increased biomass buildup derived from lipid and carbohydrate storage, this dataset suggests that OA slightly increases overall biomass of cells, but does not significantly alter their metabolite composition. Generally, light was shown to be a more dominant driver of metabolite composition than OA, increasing the relative abundances of amino acids, mannitol and storage lipids, and shifting pigment contents to accommodate increased irradiance levels. The diploid stage was shown to contain vastly more osmolytes and mannitol than the haploid stage, which in turn had a higher relative content of amino acids, especially aromatic ones. Besides the differences between the investigated cell types and the general effects on biomass buildup, our analyses indicate that OA imposes only negligible effects on E. huxleyi´s biomass composition.Owing to the hierarchical organization of biology, from genomes over transcriptomes and proteomes down to metabolomes, there is continuous debate about the extent to which data and interpretations derived from one level, e.g. the transcriptome, are in agreement with other levels, e.g. the metabolome. Here, we tested the effect of ocean acidification (OA; 400 vs. 1000 μatm CO Emiliania huxleyi is an intensively investigated microalga, especially because of its dual role in biogeochemistry. Besides being photosynthetic phytoplankton that contributes to the so-called ‘organic carbon pump’ of the oceans, it is able to produce elaborately shaped CaCO3 shells. This process of calcification alters the surrounding sea-water chemistry, driving the so-called oceanic ‘inorganic carbon pump’ [The globally distributed coccolithophore on pump’ . Also, ton pump’ .2 by the surface ocean [E. huxleyi has become a model organism for calcifying marine microalgae, not only because it is globally distributed with many ecotypes from different ocean provinces [E. huxleyi [In recent years, the global change phenomenon of ‘Ocean Acidification’ (OA) has stimulated extensive research on calcifying organisms. OA describes the consequences of the uptake of anthropogenic COce ocean , i.e., ace ocean . In the rovinces , or becarovinces –8, but arovinces , enablin huxleyi ,10 and t huxleyi , increas huxleyi , nutrien huxleyi –17 and a huxleyi –23.E. huxleyi strain RCC1216 and its haploid, non-calcifying life-cycle stage (strain RCC1217) towards OA and their modulation by light energization. It was interpreted that OA stimulated biomass production by increased photosynthesis and decreased production of calcite. The relative responses towards OA were found to be weaker under high light despite higher overall rates. In addition, the higher biomass production was sustained by less pigment per biomass, and it was concluded that under OA, cells operate at higher energy-efficiency.Previously, Rokitta and Rost  investigE. huxleyi targeting the proteome level were complicated by methodological biases. For instance, the shotgun-proteomics techniques available at date yielded merely ~100 protein identifications, and expression studies concerning the effects of OA yielded <10 significantly regulated proteins that were related to protein translation and DNA maintenance [In a subsequent study, transcriptomic analyses from these experiments were conducted using microarray-based transcriptome screenings, in which the expression of ~10.000 genes was assessed. Based on the gene expression patterns, it was interpreted that the increased biomass production likely originates from an intensified lipid metabolism, because especially genes for buildup and oxidation of lipids were found to be significantly regulated, with more synthetic machinery rather than degradative machinery being regulated. Also, genes of the oxidative branch of the pentose-phosphate pathway were found upregulated, which was taken as a supportive indication for increased lipid synthesis, as this pathway creates cytoplasmic reduction equivalents necessary for lipid synthesis . Furtherntenance ,25. DespIn addition, there is ongoing debate about consistency of interpretations derived from the newly emerging and highly diverse ‘omics’ techniques that investigate the genomes, transcriptomes, proteomes, and metabolomes of organisms . While tE. huxleyi. Because light was shown to strongly modulate the OA responses [2 (400 vs. 1000 μatm) under limiting and saturating light intensities (50 vs. 300 μmol photons m-2 s-1), thereby reproducing the experimental setup of Rokitta and Rost [To shed further light on the connections between these different abstraction layers of biological organization, we have here conducted metabolite screenings, testing the effects of OA on the metabolic composition of the diploid and haploid life-cycle stages of esponses , we accland Rost . A main E. huxleyi  were grown in sterile filtered  North Sea seawater medium, enriched with vitamins and trace metals according to the recipe for F/2 medium [3-, 100 μM) and phosphate . Cultures were grown at 15°C in a 16:8 h light/dark cycle under irradiances of 50 or 300 μmol m-2 s-1  provided by daylight lamps . All media were pre-aerated (~150 mL min-1) with air-mixtures of the desired pCO2. Air-mixtures were produced by mixing CO2-free air with pure CO2  using a mass flow controller system . The pCO2 was regularly controlled with a non-dispersive infrared analyzer system . Precultures were grown in pCO2 adjusted media for at least 7 days in exponential growth and were then used as inoculum for the main incubations. Three biological replicates of each life-cycle stage were inoculated for each setup at densities of ~2000 cells mL-1 in 2.5 L Schott glass bottles without headspace, which were then placed on a roller table (~15 rpm). The cell numbers were determined in 2–3 day intervals with a Multi-Sizer 3 particle counter (Beckman Coulter), and specific growth rates were derived [Diploid (2N) and haploid (1N) life-cycle stages of 2 medium . Medium 0), as well as directly prior to cell harvest for metabolome analyses (tfin). Calculations of the carbonate system  and silicate (7 μmol kg-1) concentrations. Calculations were based on equilibrium constants by Mehrbach et al. [Carbonate chemistry was determined in cell-free media prior to acclimations  were baprogram, . Three-ph et al. , refit bh et al.  and on dh et al. .2 and stored at -80°C. Samples were then freeze-dried before analyses to rule out degradation during transport.Cells were harvested during exponential growth phase by gentle vacuum filtration (800 mbar suction pressure) onto polycarbonate filters . For the analyses of pigments, lipids and all other metabolites, culture volumes drawn onto the filters were 300, 300, and 2000 mL, respectively. Filters were snap-frozen in liquid N5 mL of diploid cell culture were filtered onto 25 mm polycarbonate filters  and were dried and stored at room temperature. Samples were stuck on holders and sputtered with a thin conductive layer (5 nm Au/Pd 80/20) using a MED-020 sputter coater . Electron microscopic imaging was performed with an XL-30 field emission environmental scanning electron microscope .-1 flowrate and 1 μL injection volume. The column oven was set to 35°C. Eluents were acetonitrile with 5% water (A) and 0.05% acetic acid in water (B). The gradient was run from 25% B for 2 min to 60% B within 0.5 min and held for 1.3 min. Eluent B was set back to 25% within 0.2 min and the column was re-equilibrated for 3 min. D-Mannitol standard substance  was used for external calibration.Most analysis methods and the chosen instruments applied here have been described in detail in Wördenweber et al. . TherefoThe ‘fold-changes’ of all metabolites were calculated and represented in a heatmap. When a metabolite was detected in one treatment but not at all in the other, a tendency of fold-change was added manually with the value of 1.5 or -1.5 respectively. For clarity, these metabolites have been marked with a slash ‘/’.t-test was performed, and statistically significant differences were indicated by asterisks, representing p-values ≤0.05 (*) and ≤0.01 (**). All results are shown as fold-changes of mean values, with standard deviation (±SD).Results of metabolite analysis are reported either normalized per cell or normalized to biomass, using the particulate organic carbon (POC) quotas published earlier for this strain of microalgae . All datt-test , are indicated by asterisks. Results were then correlated with previous transcriptomic and proteomic findings and interpretations.Statistically significant changes in metabolite abundances between the different treatments and life-cycle stages, as determined via Student’s E. huxleyi life-cycle stages and their modulation by light, i.e. cellular energization. Results are first reported for each life-cycle stage with regard to OA responses and associated modulation by energy availability. Then, the influence of light intensity will be discussed separately, followed by differences in the life-cycle stages.We investigated the effects of OA on the metabolic composition of calcifying diploid and non-calcifying haploid 2  obtained from the earlier study by Rokitta and Rost [less abundant per unit of biomass. This is remarkable for the detected pigments, because this is in line with the decreased POC-normalized content of chlorophyll a under OA as previously observed for this strain [However, irrespective of the overall small magnitude of responses, we observed slight but consistent increases in the per-cell contents of most amino acids and some osmolytes throughout the dataset. Since these metabolite quotas are expressed per cell, the slight increases may simply reflect increases in overall organic cell mass under OA, as often observed in this and some other strains of  huxleyi ,20,46. W2 levels. High light significantly increased the cellular contents of histidine (2.7-fold), isoleucine (3.9-fold) and proline (2.6-fold), as well as mannitol (2.1-fold), the pigment h4k-fucoxanthin (2.3-fold) and TAG 9 (3.1-fold) in the low pCO2 acclimations (2. The changes were significant for chlorophyll a (1.5-fold), chlorophyll c2 (1.7-fold), 19’-hexanoyloxyfucoxanthin (hex-fucoxanthin) (2.1-fold) and also diadinoxanthin (2.1-fold). The acclimation to high light increased the cellular contents of lipids, especially when pCO2 was high, the changes being significant for most of the investigated polar lipids, i.e., C14:0 (1.7-fold), C18:1n9c (1.5-fold) and C18:4 (1.5-fold). Also triacylglycerides and alkenones showed increased cellular abundance in response to high light when pCO2 was high, as seen especially for TAG 9a (2.2-fold).High light intensity increased the abundance of many biochemicals in the diploid life-cycle stage . All fatty acids deriving from polar and non-polar lipids, except one, generally appeared to decrease in abundance, although data is lacking statistical significance. While no clear effects could be seen in the cellular contents of TAGs, OA clearly decreased abundances of all examined alkenones in haploid cells grown under high light.The biochemical composition of the haploid life-cycle stage was mostly non-responsive to OA  column cIn the low-light acclimated haploid cells, the cellular contents of most amino acids were also only insignificantly affected by OA. No general trend could be observed and only aspartate (-1.4-fold) and threonine (-1.4-fold) were significantly decreased. The abundances of all examined photosynthetic pigments slightly decreased in response to OA in the low-light treatment, although statistical significance is lacking. Most of the fatty acids derived from polar and non-polar lipids significantly decreased in abundance  and diadinoxanthin (1.4-fold). All examined fatty acids were generally increased in abundance in response to high light, with significant changes in the cellular contents of the polar lipids C14:0 (1.5-fold), C16:0 (1.5-fold), C18:0 (1.6-fold) and C18:4 (1.5-fold). Alkenones significantly increased in cellular abundance, with fold-changes ranging between 1.5- and 8-fold.In cells of the haploid life-cycle stage that were grown under low pCO2, but none of these small changes was significant. Similarly, high light did not significantly affect the contents of fatty acids derived from polar and non-polar lipids. In contrast, high light increased alkenone contents of cells acclimated to high pCO2, with significant effects being determined for the alkenone C38:3 Me , tryptophan (from -20.53 to -87.61) and tyrosine (from -19.26 to -60.77). The high contents of aromatic amino acids in the haploid stage might suggest a higher activity of the shikimate pathway that also produces precursors for, e.g., mycosporine-like amino acids [The comparison of the two life-cycle stages  that showed some differences as well as similarities. We observed partially large differences in metabolite concentrations between the present experiment and Wördenweber et al. [We used the same microalgal strains, cultivation techniques and methods as previously presented in Wördenweber et al. . Differer et al. , with thr et al. , about hThe diploid life-cycle stage reproducibly contained vastly more DMSP than the haploid stage, although the specific fold-changes vary greatly between this study and Wördenweber et al. . This isThe higher abundance of mannitol in the diploid stage as well as the higher abundance of citrate in the haploid stage were closely reproduced between the two studies and also most other small carboxylic acids were detected to have similar distributions. Also the stage-specific distributions of pigments and alkenones were alike in both experiments. In contrast, the polar and neutral lipid concentrations as well as the contents of TAGs differ between the studies for almost all detected compounds, indicating that the cellular composition of lipids is subject to great biological variability. In contrast to Wördenweber et al. , we founMausz and Pohnert  also comin-vivo metabolome’, the analyses of biochemical compounds after treatment, e.g. hydrolysis of (un)polar lipids, suggest that OA has a negligible effect on the overall composition of biomass and does not alter the contribution of lipophilic compounds. Based on our data, no statements on the abundance of glucans can be made, but the development of analytical protocols for this diverse and somewhat cryptic class of biopolymers should be a priority for future studies. Whereas profound emergencies like nutrient starvation severely affect the physiology of the cells [2 and concomitant acidification seem to only slightly modulate the steady state fluxes between the assessed metabolite pools. The rearrangements in the biochemical pathways of plastidary and lipid metabolism that were previously postulated based on transcriptomics were often indicated, but their order of magnitude was either too small to be resolvable or masked by the comparably large biological variance, underlining the flexibility of cellular biochemical networks. Some trends deduced from the transcriptome studies could be reproduced, namely overall increased biomass production and shifts in pigmentation [The obtained biochemical data show that OA, while significantly affecting the transcriptomes of haploid and diploid life-cycle stages , has farhe cells ,36, highentation . The appS1 Table(XLSX)Click here for additional data file.S2 TableAlkenone-derived temperature signals.(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 TableConcentrations and fold-changes of metabolites.(XLSX)Click here for additional data file.


Lactobacillus (Lb.) paracasei IJH-SONE68 produces a novel exopolysaccharide (EPS). Furthermore, we have shown that the EPS inhibits the catalytic activity of hyaluronidase (EC 3.2.1.36) promoting inflammatory reactions. To evaluate the anti-allergy and anti-inflammatory effects of the EPS, in the present study, we employed the picryl-chloride-induced delayed-type (type IV) allergy model mice, which is used to evaluate the contact dermatitis. Oral administration of the EPS was observed to reduce the ear swelling in the model mice. We also observed that the overexpression of ear interleukin-4 (T helper (Th) 2 cytokine) mRNA and the increase in serum immunoglobulin E (IgE) are repressed. However, the expression of interferon-γ (Th1 cytokine) was not accelerated in all of the allergen-challenged model mice. The improvement may be responsible for the Th2 downregulation rather than the Th1 upregulation. In addition, the symptom of immediate-type (type I) allergy model mice was improved by oral administration of the IJH-SONE68 cell (data not shown). We can conclude that the IJH-SONE68-derived EPS is useful to improve the type I and IV allergies including atopic dermatitis.Allergic disease is one of the most important and common health problems worldwide. We have previously demonstrated that a fig leaf-derived lactic acid bacterium  Lactic acid bacteria (LABs) are typical probiotics that are an order of gram-positive and generally non-pathogenic bacteria. Probiotics are defined as living microorganisms that confer health benefits to hosts such as humans .Lactobacillus (Lb.) delbrueckii subsp. bulgaricus  is orally administrated, the bacterium inhibits the growth of intestinal toxin-producing bacteria [LABs produce a large amount of lactic acid, sometimes together with acetate or ethanol . Some LAbacteria . That hybacteria ,6,7,8.Lb. paracasei IJH-SONE68, isolated from a fig leaf, produces neutral and acidic exopolysaccharides (EPSs) [N-acetylglucosamine (GlcNAc), demonstrating that the neutral EPS has a novel structure that has never been reported [We have isolated many kinds of plant-derived LABs, which will be useful for preventive medicine ,10,11. Is (EPSs) . In the reported . On the reported .Hyaluronidase catalyzes the hydrolysis of hyaluronic acid as a main constituent of the extracellular matrix. The enzyme promotes inflammation by hydrolyzing hyaluronic acid under inflammatory conditions ,14,15,16Picryl chloride (PiCl), which has been utilized for a long time, is a simple chemical hapten reagent  used to p < 0.01) repressed the thickness of the swollen ear on the PiCl-induced delayed-type allergy model mice, whereas the administration of sterile distilled water did not. Although the observed repression was not significant in the 10- or 100-fold diluted fermented pineapple juice group, the swelling was definitely decreased when compared with the positive control group. Understandably, the pineapple juice without fermentation did not repress the ear swelling.In this study, pineapple juice supplemented with sake lee was used as the culture medium for the IJH-SONE68 strain. 12 cfu/mL and the EPS solution (1 mg/mL) were employed. Oral administration of the heat-killed cells significantly repressed the inflammatory reaction, whereas that of the living cells did not, suggesting that the IJH-SONE68-derived EPS is the functional molecule with regard to delayed-type allergy. We also examined whether the functional molecule is the neutral or acidic EPS (1.0 or 0.1 mg/mL solution). In a subsequent experiment, we evaluated whether the LAB cells and EPS fraction  prevent or repress the PiCl-induced delayed-type allergy B. Under To understand the preventive mechanism against the contact dermatitis model observed with administration of the IJH-SONE68-derived EPSs, the mRNA expression levels of T helper (Th) 1-type (interferon (IFN)-γ) and Th2-type (interleukin (IL)-4, 5, and 13) inflammatory cytokines in ear tissue were measured using quantitative reverse transcriptional (qRT)-PCR analysis. The IgE level in the serum of type I allergic patients, which are known as pollinosis and allergic rhinitis, is significantly higher than in that of healthy persons . In the The paraffin-embedded ear tissue sections from each mouse were subjected to histological comparison . As a rep = 0.077 and 0.094, respectively) the ear thickness.To confirm whether the EPS has a therapeutic effect on the delayed-type allergy model mice, we tested direct application of the IJH-SONE68-derived EPS to the mouse earlobe . The resLb. paracasei, which has been used to manufacture dairy products, have been reported to be effective for the immune-modulation and the reduction of allergic symptoms. With respect to the health benefits, same species have been reported as follows: Lb. paracasei MCC1849 enhances the expression of antigen-specific IgA [Lb. paracasei LP33 is effective against allergic rhinitis [Lb. paracasei KW3110 reduces allergic inflammation in patients with cedar pollinosis [Several strains of ific IgA , Lb. parrhinitis , and Lb.llinosis . We demoThe type I allergy, which is an immediate allergy, includes pollinosis and allergic rhinitis as typical disorders . In thisThe type IV allergy, which is a delayed allergy, is thought to be mainly associated with activation of the Th1 cell. If the antigen is present, the proliferated Th1 cell releases cytokines to induce granulocyte infiltration and the accumulation of macrophages. PiCl is an agent that confers the picryl group to skin proteins . The PiCIn the IFN-γ or IFN-γ receptor–deficient mice, the contact dermatitis allergic reactions were also observed as in the wild-type mice ,28,29,30In general, the observed improvement of allergic symptoms upon oral administration of the bacterial cells is due to the inhibition of Th2 cell proliferation via Th1 cell induction by antigen-presenting cells that recognize the bacterial cell as an antigen . By thisLb. delbrueckii subsp. bulgaricus OLL1073R-1 has been reported to enhance the natural killer (NK) cell activity in mice [Lactococcus (Lc.) lactis subsp. cremoris GCL1176 [Lc. lactis subsp. cremoris FC-derived EPS observed on the dermatitis model mice seems to be due to the modulation of the systemic immune system through a Peyer’s patch-mediated mechanism [Since EPS is composed of a long chain of monosaccharide units, the macromolecule does not seem to be absorbed into the blood through the intestinal cell wall without digestion by intestinal microorganisms and enzymes. The EPS produced by  in mice . The sti in mice . In the  in mice . Tohno e GCL1176 . Judging GCL1176 ,40. On techanism . Howeverechanism ,43. TherIn the present study, the IJH-SONE68-derived EPSs are found to exhibit anti-inflammatory activity by direct application to the mouse ear. The predicted mechanism may be distinguished from the improvement observed with oral administration. The EPS applied to the skin is predicted to permeate the skin, and the macromolecule may inhibit the enzymatic activity of hyaluronidase, which stimulates the inflammatory reaction. In the previous study, we evaluated the hyaluronidase-inhibitory activity of several LAB-derived EPSs, sodium cromoglicate, dipotassium glycyrrhizinate, and ketotifen fumarate, which are known as substances repressing the type I allergy ,44,45. AIn the previous study , the maiBifidobacterium are known to confer health benefits to humans. In addition to the bacterial cells, LAB-derived EPS provides some health benefits [LABs and a genus benefits ,44,45,50benefits , and enhbenefits  of EPSs benefits , to exhibenefits , and to benefits . Recentlbenefits .Lb. paracasei IJH-SONE68. A modified semi-defined medium (SDM) supplemented with a vitamin mixture (0.2 v/v %) and trace element solution (0.1 v/v %) instead of yeast nitrogen bases was used as a main culture medium [A de Man, Rogosa, and Sharpe (MRS) medium  was used as a seed culture medium for e medium ,56,57.12 cfu/mL. If necessary, the washed cell mass was heat-treated in an autoclave (at 121 °C for 20 min). Prior to cultivation of the LAB strain using pineapple juice supplemented with 1 (w/v) % spray-dried sake lees, the culture medium was adjusted to pH 6.0 with NaOH and sterilized at 100 °C for 10 min. If necessary, the pineapple juice fermented by the IJH-SONE68 strain was 10- or 100-times diluted by sterile distilled water.The IJH-SONE68 cell mass used for oral administration was prepared by the following method: the LAB strain was grown in the modified SDM at 28 °C for 48 h. The cell mass, which was obtained by centrifugation of the cultured broth, was washed and diluted with sterile distilled water to 1 × 10v/v) volume of trichloroacetic acid (TCA) solution was added to the culture broth. The culture supernatant obtained by centrifugation was added with the same volume of acetone. The resulting precipitate containing the EPS was dissolved in a 50 mM Tris–HCl buffer (pH 8.0). After treatment with nuclease and protease, the neutral and acidic EPSs were separated by column chromatography using the anion exchange resin  as described previously [The stock culture of the IJH-SONE68 strain stored at −80 °C was inoculated into a fresh MRS medium. After the two-day standing cultivation at 28 °C, the cells collected by centrifugation were washed with a sterilized phosphate-buffered saline (PBS: pH 7.4) and resuspended into the same volume of PBS. The resulting cell suspension was inoculated into a modified SDM with a 0.2% volume and grown at 28 °C for 2 days. After the cultivation, at a final concentration, a 4%  BALB/cAJcl seven-week-old mice were purchased from CLEA Japan, Inc. , and divided into 4–6 experimental groups of 5 mice each after a 1-week acclimation period. The mice were housed in plastic cages under 20–26 °C, 40–60% humidity, and 12-h light/12-h dark conditions. The mice were freely fed with an MF diet  and could freely access water. The animal experiment was conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals of Hiroshima University. The mice were distinguished by marking their tails with different colors using felt markers . After the experimental period, the mice were euthanized via inhalation of anesthesia with isoflurane. The experimental protocol for the animal study was approved by the committee of the Research Facilities for Laboratory Animal Science of Hiroshima University .w/v) PiCl dissolved into an acetone: sunflower oil mixture (4:1). In this experiment, a 20-μL portion of the 7% (w/v) PiCl solution was applied to each footpad of the mice. One hundred microliters of the PiCl solution was also applied to the breasts and backs of the mice. One week later, 20-μL of 1% (w/v) PiCl solution was applied to both the front and back of the ear. The thickness of ear was measured just before and 24 h after the allergen challenge using a vernier scale (resolution 0.02 mm), and the averaged increment of ear thickness (Δ ear thickness) in the negative control group was only 0.0056 mm, showing that the value is much smaller than the measurement limit (0.02 mm). Therefore, we conclude that the employment of the PiCl-induced mice model is suitable to evaluate the anti-hypersensitive effects of samples. The Δ ear thickness was assessed using the Tukey–Kramer test [The after a one-week acclimation period, we began orally administering a 200-μL aliquot of each sample to mice every day during the experimental period using oral feeding needle. After 6 days, the mice were immunized with 7%  was applied to the back and front surfaces of the ears of mice without an oral administration phase. The ear thickness was measured after an additional 20 h of breeding. The animal experiment is summarized in The EPS samples were dissolved into sterile distilled water. If necessary, the EPS samples and the fermented pineapple juice were diluted with sterile distilled water.Total RNA was extracted from each mouse ear using NucleoSpin RNA II  according to the manufacturer’s instruction manual. The RNA sample was reverse transcribed using the ReverTra Ace qPCR RT master mix with gDNA remover  according to the manufacturer’s instruction manual. The qRT-PCR was conducted on the PikoReal real-time PCR system  using the KAPA SYBR Fast qPCR Kit . The qPCR was conducted under the following conditions: an initial 30 s at 95 °C, followed by 40 cycles of 5 s at 95 °C and 30 s at 60 °C. The relative transcriptional levels, which were normalized to a reference gene (β-actin), of the IL-4, 5, 13, and IFN-γ were analyzed using the ΔΔCT method. The primers used in this experiment are as follows: 5′-GGGACGACATGGAGAAGA-3′ and 5′-CATACAGGGACAGCACAG-3′ for β-actin; 5′-TCTCGAATGTACCAGGAGC CATATC-3′ and 5′-AGCACCTTGGAAGCCCTACAGA-3′ for IL-4; 5′-GAGAAGTG TGGCGAGGAG-3′ and 5′-GGGGTTTTTGCATCTGT-3′ for IL-5; 5′-GGAGAGGCA GGGAGGAGG-3′ and 5′-GTGGGGTGGGGGTGTTG-3′ for IL-13; and 5′-GAGGAA CTGGCAAAAGGATG-3′ and 5′-GCTGATGGCCTGATTGTCTT-3′ for INF-γ.Serum was extracted from blood samples collected from the mice of each group using a Separapid microtube S . The IgE concentration in each serum was measured using the Mouse IgE ELISA development kit (HRP) , according to the manufacture’s instruction manual.v/v) formalin and embedded in a paraffin block. The histopathological tissue section was stained with hematoxylin-eosin (HE).Mouse ears were obtained 24 h after the allergen challenge. The ears were fixed with 10% (w/v) glutaraldehyde for 2 h at room temperature. The PBS-washed cells were used for SEM analysis. The SEM image was observed and captured at a magnification of 12,000×, with an acceleration voltage of 15 kV, using a Hitachi Tabletop Microscope TM3030Plus .The IJH-SONE68 cells were fixed with 2% (It has been known that IgE worsens the symptoms of atopic dermatitis as a mixed model of the type I and IV allergies. In the present study, we observed that the oral administration of the IJH-SONE68-derived EPS represses the expression of IL-4 and serum IgE accelerated in the PiCl-induced delayed-type allergy model mice, suggesting that the IJH-SONE68-derived EPS is effective to improve both types of allergic reactions. Significantly, the acidic EPS is more effective than the neutral one. The improvement may be responsible for the Th2 downregulation rather than Th1 upregulation. It is interesting and significant to understand the relationship between the chemical structure and physiological functions of the EPS.


Geophila repens (L.) I.M. Johnst, a perennial herb, belongs to the Rubiaceae family. In this study, we identified the chemical composition of the Geophila repens essential oil (GR-EO) for the first time. Totally, seventy-seven compounds were identified according to GC and GC-MS, which represent 98.0% of the oil. And the major components of GR-EO were β-caryophyllene (23.3%), β-elemene (8.0%), farnesyl butanoate (7.4%), myrcene (3.5%), and trans-nerolidol (3.3%). Then we evaluated the antibacterial activities of GR-EO and the synergistic effects of GR-EO in combination with commercial antibiotics using the microdilution and Checkerboard method. The results demonstrated that GR-EO possessed an excellent broad spectrum antibacterial activity, especially against Pseudomonas aeruginosa and Bacillus subtilis. It also showed that the combined application of GR-EO with antibiotics led to synergistic effects in most cases. And the most prominent synergistic effect was noticed when GR-EO was in combination with Streptomycin and tested against Escherichia coli  of 0.13). Additionally, the results of a Griess assay revealed that GR-EO exhibited a potent inhibitory effect on NO production in lipopolysaccharide (LPS)-activated RAW 264.7 (murine macrophage) cells. In conclusion, the combination of GR-EO and the commercial antibiotics has significant potential for the development of new antimicrobial treatment and reduction of drug resistance. At present, antibiotic resistance is one of the greatest threats to public health, so it is clear that searching for new antibacterial agents is absolutely necessary. Antibiotic resistance can be intrinsic or acquired and can be transmitted within the same or different species of bacteria. The mechanisms can be divided into different categories: (i) modification of the active site of the target resulting in a reduction in the efficiency of binding of the antibiotic; (ii) direct destruction or modification of the antibiotic by enzymes produced by the bacterium; (iii) efflux or removal of antibiotic from the cell cause a reduced amount of antibiotic; or (iv) production of an alternative target that is resistant to inhibition by the antibiotic (metabolic by-pass) ,2,3.Exploring the natural products from plants could become an interesting alternative therapy ,4,5. SomRubiaceae family contains about 13,500 species and 611 genera. It consists of terrestrial trees, shrubs, lianas, and herbs. Rubiaceae has a cosmopolitan distribution. However, the largest species diversity is concentrated in the tropics and subtropics [Geophila is a small genus of the Rubiaceae family, containing about 30 species. It is found primarily in Asia-Tropical and Africa.The btropics . GeophilGeophila repens (L.) I.M. Johnst belongs to the Rubiaceae family and is widely distributed in the tropics and subtropics of Africa, Asia, through the Pacific to the Americas and Caribbean. It is a creeping, evergreen, perennial herb growing up to 10 cm tall and often forming dense close colonies in shaded places. The leaves of Geophila repens are sometimes chewed to remedy coughs. And a decoction of the boiled plant is also used as a treatment for coughs. The fruits of Geophila repens are considered to be an effective antifungal agent [al agent .Geophila repens essential oil (GR-EO) by hydrodistillation, and identified the chemical composition of GR-EO using GC and GC-MS for the first time. Then we demonstrated the in vitro antibacterial properties of GR-EO against four bacterial strains by disc agar diffusion and broth microdilution methods, and studied the synergistic effect of the combination of GR-EO with standard antibiotics using Checkerboard method. In addition, we evaluated the inhibitory effects of GR-EO on NO production in lipopolysaccharide (LPS)-activated murine macrophage RAW 264.7 cells.In the present study, we extracted the Geophila repens are listed in trans-nerolidol (3.3%), and α-caryophyllene (3.3%).The components of the essential oil with their retention indices (RI) from the S. aureus and B. subtilis) and two gram-negative bacteria (E. coli and P. aeruginosa). The antibacterial activities of GR-EO were qualitatively assessed by the presence or absence of inhibition zone diameters (DIZs). The results are presented in P. aeruginosa, B. subtilis, and S. aureus, respectively. However, this oil showed low activity (DIZ < 7.5 mm) towards E. coli.GR-EO was tested against four pathogenic bacteria, including two gram-positive bacteria  and B. subtilis , followed by S. aureus . However, it showed a weak activity against E. coli .More accurate data on the antibacterial activity were obtained through the MIC and MBC  values of the oil. From the data, it is evident that GR-EO displayed a broad spectrum antibacterial activity against the tested bacterial strains. As shown in The evaluations of synergistic effects of GR-EO in combination with four selected conventional antibiotics were determined using the Checkerboard method. The interaction data in the form of the fractional inhibitory concentration indices (FICIs) are listed in B. subtilis (FICI of 0.38), P. aeruginosa (FICI of 0.38), and E. coli (FICI of 0.5). While an indifference effect was observed in S. aureus (FICI of 1.5).Synergistic effects of Chloramphenicol combined with GR-EO according to the calculated FICIs are presented in E. coli (FICI of 0.13), P. aeruginosa (FICI of 0.38) and S. aureus (FICI of 0.38) when there was a combination of GR-EO with Streptomycin, while an additive effect was seen in B. subtilis (FICI of 0.75).According to the obtained results presented in P. aeruginosa (FICI of 0.25) and E. coli (FICI of 0.5) according to S. aureus and B. subtilis were not performed because these strains were sensitive when antibiotic was administered alone.Combination of GR-EO and Penicillin displayed synergistic activity against both P. aeruginosa (FICI of 0.38) and an additive effect against E. coli (FICI of 1). The combination against strains of S. aureus and B. subtilis were not performed because these strains were sensitive when antibiotic was administered alone.As is presented in We determined the inhibitory effect of GR-EO on the NO production of RAW 264.7 macrophages using Griess reagent system. As shown in trans-nerolidol (3.3%), and α-caryophyllene (3.3%). It is noteworthy that some major compounds had been reported to have various bioactivities. Such as β-caryophyllene, a natural bicyclic sesquiterpene, which is a constituent of many essential oils, especially clove oil. Previous studies have indicated that β-caryophyllene possessed a significant activity against gram-positive bacteria with MIC values from 0.032 to 0.256 mg/mL [In the present study, the major constituents in the GR-EO were found to be β-caryophyllene 23.3%), β-elemene (8.0%), farnesyl butanoate (7.4%), myrcene (3.5%), 56 mg/mL . Further56 mg/mL ,21. Anot56 mg/mL ,23,24..3%, β-elAccording to the results of antimicrobial activity testing, GR-EO had a potent effect on both gram-negative and gram-positive bacteria. The mechanisms of the antimicrobial action of essential oils are still not completely understood. Some studies suggested that, the amphiphilic nature of terpenoids due to the presence of hydrophobic skeleton and hydrophilic functional groups enables easy transport of these phytochemicals across biological membranes of microbial cells, thereby allowing them to reach cytosol and interact with key intracellular biomolecules ,25. GeneB. subtilis, S. aureus, P. aeruginosa, and E. coli. The present study revealed the synergistic effect of GR-EO in combination with Chloramphenicol, Streptomycin, Penicillin, or Ampicillin against a wide range of bacterial strains. It is to be noted that remarkable synergistic action was observed against E. coli by the combination of GR-EO with Streptomycin (FICI of 0.13). Escherichia coli, a gram-negative bacterium, has been linked to numerous common bacterial infections, including bacteremia, cholecystitis, cholangitis, urinary tract infection (UTI), and other clinical infections such as neonatal meningitis and pneumonia [E. coli with the FICI values of 0.5.Furthermore, the synergistic effects of GR-EO in combination with four selected conventional antibiotics were evaluated against neumonia . TherefoP. aeruginosa (FICI range of 0.25 to 0.38). Pseudomonas aeruginosa, a gram-negative bacterium, is one of the leading causes of nosocomial infections. Severe infections, such as pneumonia or bacteremia, are associated with high mortality rates and are often difficult to treat, as the repertoire of useful anti-pseudomonal agents is limited; moreover, P. aeruginosa has high intrinsic resistance to antibiotic and exhibits remarkable ability to acquire resistance to these agents during treatment [P. aeruginosa is significantly due to low permeability of the cell wall, which restricts the uptake of antibiotics, and the genetic capacity to express a wide repertoire of resistance mechanisms, like efflux pumps and enzymes, which modify or degrade antibiotics and drug targets [From the results obtained, what is more noteworthy is that the combinations of GR-EO plus all tested antibiotics have conspicuous synergistic effects when tested against reatment . The abi targets . TherefoB. subtilis (FICI of 0.38), and with Streptomycin against S. aureus (FICI of 0.38). Staphylococcus aureus is a major human pathogen that causes a wide range of clinical infections. It is a leading cause of bacteremia and infective endocarditis as well as osteoarticular, skin and soft tissue, pleuropulmonary, and device-related infections [S. aureus infections could include combined use of GR-EO and Streptomycin.Additionally, synergy was also noted for another two interactions, namely GR-EO in combination with Chloramphenicol against fections . TherefoThere are fewer reports on the mode of action of the essential oils in combination with antibiotics. And some mechanisms of antimicrobial interaction that produce synergism are generally accepted, such as the sequential inhibition of a common biochemical pathway, inhibition of protective enzymes, and use of cell wall active agents to enhance the uptake of other antimicrobials .This study indicates that the combination of GR-EO and the commercial antibiotics has significant potential for the development of new antimicrobial treatment and reduction of drug resistance, which will permit to find the treatment of several infections caused by microorganisms.Additionally, we also evaluated the inhibitory effects of GR-EO on NO production in lipopolysaccharide (LPS)-activated murine macrophage RAW 264.7 cells. LPS is a major component of the outer membrane of gram-negative bacteria, and it has been recognized as important in determining both the virulence of these organisms and the symptomatology that accompanies gram-negative bacteria infection . When batrans-nerolidol, which have been reported to possess excellent inhibitory effect on the NO production by LPS-stimulated macrophages [The evaluation of NO production showed that GR-EO presented a significant inhibitory effect. It could be attributed to the presence of significant amounts of β-caryophyllene and rophages ,38. Addirophages .Geophila repens was collected in July 2016 from Guangxi Province of China, and was identified by Associate Prof. Hong Zhao of Shandong University. A voucher specimen (No. 10807) was deposited at the Laboratory of Botany of Marine College, Shandong University.w/w). The oil was stored at 4 °C in dark vials until analysis.The fresh plant material (600 g) was hydrodistilled for five hours using a Clevenger apparatus to extract GR-EO  using an Agilent 6890  equipped with flame ionization detector . GC-FID analysis of the oil was carried out on HP-5 MS capillary column  under the following conditions: Program the oven temperature stay at 60 °C for 1 min, then heat to 280 °C at a rate of 6 °C/min, hold at 280 °C for 2 min. The carrier gas used was helium at a constant flow rate of 1.2 mL/min, while the detector and injector temperatures were 250 °C and 240 °C, respectively.m/z 50 to 550.The GC-MS analysis was carried out on Hewlett Packard 6890 gas chromatograph (Agilent) attached to a HP-5MS fused silica column, interfaced with a Hewlett Packard 5975C mass selective detector operated by HP Enhanced ChemStation software (Agilent). The analytical conditions for GC were the same as those mentioned for GC-FID. The injection volume was 0.2 μL of 1% solution prepared in n-hexane with split ratio 1:50. Mass spectra were acquired in EI mode at 70 eV. The mass range was from 7–C30 n-alkanes, under the same experimental conditions), and computer matching with Nist MS Search 2.2 Mass Spectral Database for GC-MS as well as comparisons of their mass spectra with those of authentic samples or with data already available in the literature. The result of the analysis is shown in Identification of the constituents was performed on the basis of their retention time, retention indices  and two gram-negative  bacteria. All strains were cultured at 37 °C on Mueller-Hinton medium.The antibacterial activity of the investigated essential oil was tested against four bacteria strains. The following microorganisms were used in this work: two gram-positive (8 Colony-Forming Units (CFU)/mL using a 0.5 McFarland turbidity standard, after that, the suspension was diluted to obtain an inoculum of 106 CFU∙mL−1.Each strain was grown in liquid nutrient broth at 37 °C for 18 h before being used for antimicrobial tests. The microbial load was adjusted to 106 CFU/mL. Then 100 μL of bacterial suspension was spread on the MHA plates, on which filter paper discs (6 mm in diameter) individually saturated with essential oil  were placed. The plates were incubated at 37 °C for 24 h. The DIZ values were measured with a caliper in millimeter. Chloramphenicol (10 μg/disc) was used as the control. All the tests were repeated in triplicate.Antibacterial activities were assessed by the disc agar diffusion method recommended by CLSI  (2012) . BrieflyThe MIC values were performed in 96 well-microplates using the microdilution assay according to the literature previously described by Ellof (1998) with slight modifications . The essFor the determination of the MBC, a sample of 100 μL from each well  was subcultured on the MHA plates and incubated at 36 °C for 18–24 h (overnight). The MBC is defined as the lowest concentration without any bacterial growth.The synergistic effect of triterpenoids in combination with antibiotics was determined using the Checkerboard dilution method . Accordi2 and humidified air atmosphere.The murine macrophage cell line RAW 264.7 was cultured in DMEM (Dulbecco’s modified eagle medium) supplemented with 10% FBS, 100 U/mL of penicillin and 100 μg/mL of streptomycin. All cells were grown at 37 °C in 5% CO3 cells per well) with 200 μL of culture medium were seeded in 96-well plates and grown for 24 hours to allow cell attachment. After that, the cells were treated with or without the dilutions of the oil for 24 h. Then, 20 μL MTT solutions (5 mg/mL in PBS) were added to each well for another 4 h and the resulting crystals were dissolved in dimethyl sulfoxide (DMSO). The optical density was read at 570 nm. Cytotoxicity was calculated from the plotted results using untreated cells at 100%. The percentages of cell growth were calculated as follows:The cytotoxicity of GR-EO was evaluated by MTT assays. GR-EO was solubilized in DMSO, and afterwards diluted with culture medium for use. RAW 264.7 cells (5 × 104 cells/mL) and co-incubated with samples and LPS (10 μg∙mL−1). After incubation at 37 °C for 24 h, each 50 μL of the culture supernatant was mixed with 50 μL of Griess reagent I and 50 μL of Griess reagent II at room temperature. Absorbance was measured at 540 nm using a microtiter plate reader. A series of known concentrations of sodium nitrite (NaNO2) was used as the standard curve.To investigate the anti-inflammatory activity of GR-EO, NO production was assessed according to the Griess reaction . Each tet-test. Differences were considered significant at p ≤ 0.05. GraphPad, Prism version 7.0 was used for statistical analysis.All of the tests were conducted in triplicate and the experimental data were expressed as means ± SD. Statistical analysis was performed by Student’s P. aeruginosa and B. subtilis. Additionally, synergistic effects were observed against tested bacteria when GR-EO was combined with conventional antibiotics, which could lead to new options for the treatment of infectious diseases caused by bacteria and emerging drug resistance. Moreover, no antagonistic effect was observed in this study further augments the potential for the use of GR-EO in combination with antibiotics.The current study identified the chemical components of GR-EO by GC and GC-MS, evaluated the antibacterial activity of GR-EO alone and the synergistic effects of the combinations between GR-EO and antibiotics, and assessed the NO production inhibitory effect. The results obtained revealed that GR-EO exhibits potent inhibitory effect on LPS-induced NO production in RAW 264.7 macrophages, and possesses significant antibacterial effect, especially against


A post hoc analysis was performed to assess the long-term effect of azithromycin on FEV1, BOS progression and survival .Azithromycin stabilises and improves lung function forced expiratory volume in one second  to assess FEV1 in the placebo group improved after open-label azithromycin and was comparable with the treatment group by 6 months. FEV1 decreased after 1 and 5 years and was not different between groups. Patients (n=18) with rapid progression of BOS underwent total lymphoid irradiation (TLI). Progression-free survival (log-rank test p=0.40) and overall survival (log-rank test p=0.28) were comparable. Survival of patients with early BOS was similar to late-onset BOS (log-rank test p=0.74).FEV1 decline. BOS progression and long-term survival were not affected by randomisation to the placebo group, given the early cross-over to azithromycin and possibly due to TLI in case of further progression. Performing randomised placebo-controlled trials in lung transplantation patients with BOS with a blinded trial duration is feasible, effective and safe.Long-term treatment with azithromycin slows down the progression of BOS, although the effect of TLI may affect the observed attenuation of FEV Placebo-controlled trials in patients with bronchiolitis obliterans syndrome (BOS) are scarce, and long-term outcomes of such trials are generally lacking in lung transplantation.Long-term treatment with azithromycin slows down the progression of BOS; BOS progression and long-term survival are not affected by randomisation to the placebo group.This post hoc analysis strengthens the evidence of long-term azithromycin treatment in BOS and shows the validity and ethical appropriateness of conducting placebo-controlled trials in this difficult area.Lung transplantation is a preferred option in selected patients with end-stage lung disease that continues to progress.1) <80% maximum recorded post-transplant (BOS grades 1–3) and ability to give informed consent. Exclusion criteria were airflow limitation caused by other clinical problems , known hypersensitivity to azithromycin or any of the macrolide antibiotics, participation in another drug-related trial within 90 days, severe renal or hepatic impairment and use of concomitant medication that would interact with azithromycin. After the study period of 12 weeks, all patients received open-label azithromycin. Patients were followed up in the outpatient clinic on a regular basis (3–4 months) with lung function, FEV1 and forced vital capacity (FVC) until 6 years after inclusion of the last patient. There was one patient lost to follow-up originally allocated to the azithromycin group. Data of 45 patients were available for post hoc analysis. A flowchart is shown in Patients were initially included for the double-blind, randomised placebo (n=23) controlled trial with azithromycin 250 mg (n=23) on alternate days  between November 2006 and December 2010 from the Freeman Hospital NHS, Newcastle upon Tyne, UK. Inclusion criteria were male or female lung transplant recipients over the age of 16 years, airflow limitation–forced expiratory volume in one second . All patients provided written informed consent before randomisation. The institutional review board approved the study.All patients received standard triple immunosuppression with calcineurin inhibitor (cyclosporine A or tacrolimus), a cell cycle inhibitor (azathioprine or mycophenolate mofetil) and prednisolone 0.1 mg/kg. Patients with rapid progression of graft dysfunction despite azithromycin during the open-label phase, defined as a persistent or progressive decrease in FEV1 at follow-up or referral for TLI, survival and the effect of BOS severity and time to BOS were assessed by construction of Kaplan-Meier curves and log-rank test. A p value of <0.05 was regarded as statistically significant.GraphPad Prism V.8.0 software  was used for statistical analyses, all performed on an intention-to-treat basis by comparing patients assigned in the initial study to the placebo and azithromycin groups. Data are shown as median with IQR. Two-way analysis of variance was used to analyse the evolution of lung function. Freedom from progression of BOS, defined as >10% drop in FEVPatient and public involvement statement: not involved.Data of 45 patients were available for analysis. Follow-up time was not different between the groups. (azithromycin 11.0±4.2 years vs placebo 9.0±4.2 years). 1 litres in the patient groups after open-label azithromycin. After 6 months, FEV1 of the initial placebo group increases but is not different from the patients randomised to the azithromycin group (placebo: 1.7 L (1.5–2.6) vs azithromycin: 1.5 L (1.1–2.3), p=not significant (NS)). After 1 year  vs azithromycin; 1.5 L (1.2–2.3), p=NS) and 5 years, FEV1 decreases gradually (placebo: 1.4 L (0.9–1.9) vs azithromycin: 1.6 L (1.3–2.4), p=NS) but is not significantly different between the groups.1 (8 patients in the placebo group and 10 patients in the azithromycin randomised group). The time from start of azithromycin to TLI was 7.7 months (0.3–36.5) and was not different between the groups. After the start of the randomised trial, four patients randomised to placebo received rescue azithromycin because of rapid deterioration as per protocol, and one patient withdrew consent.During the follow-up period, 24 patients died as a consequence of respiratory failure or infections. There was no effect of initial randomisation on survival . In addiThis work represents the first post hoc analysis of the long-term effect of azithromycin in BOS of a double-blind placebo-controlled trial in lung transplantation patients. Compared with the group initially treated with azithromycin, the lung function of patients on placebo stabilises after starting azithromycin. Progression-free survival and overall survival were not different between the groups. In addition, in this group, BOS severity and the time to development of BOS did not affect survival. This analysis shows that conducting randomised placebo-controlled trials is safe in terms of progression of BOS and long-term survival.et al1 with azithromycin.et al.1 in the first 6 months. The data in our study showed that in the treatment group, lung function stabilises, and in the placebo group, after an initial decline, lung function improves after 3 months of open-label treatment with azithromycin to a level similar to the treatment group. Although the lung function decline is progressive over time in our study group, this is a gradual process. In the study by Lama et al,et al data, there was no progressive decline in lung function with azithromycin in our study group in the first year after BOS onset, and in agreement with the observational study by Shitrit et al,1; however, an approximately 0.30 mL (10%) decline in FEV1 in 4 years with azithromycin seems a clinical significant finding and supports the positive impact of azithromycin.A recent meta-analysis by Kingah 1 or referral for TLI, was not different between the groups. This might suggest that performing a randomised placebo-controlled trial is safe in terms of progression of BOS. In addition, long-term survival was not different between the groups. Our study showed a better 5-year survival when compared with the overall survival in the study by Finlen Copeland et al.et al study, where earlier initiation of azithromycin was protective of BOS progression and death.et al.Progression of BOS, arbitrarily defined as >10% decline of FEVLimitations of this report include the retrospective design, being a single-centre study and the size of the study population. However, the study population reflects an average patient population in a lung transplant centre, and we believe the conclusions are both valid and important.1 decline. Importantly, BOS progression and long-term survival are not affected by randomisation to placebo for a 3-month period, given the early cross-over to azithromycin and possibly due to TLI in case of further progression. Survival was not different between early or late onset of BOS and was better than many previously reported series.In conclusion, long-term treatment with azithromycin initially improves lung function then slows down the progression of BOS, although the effect of TLI may have affected the observed attenuation of FEV


Some amino acids have been found to play critical roles in affording thermotolerance. Recently, it was found that in ovo on embryonic day 7 to determine their effect on rectal temperature (RT), body weight (BW) and thyroid hormones at hatching. Changes in RT, BW, and thermotolerance in post-hatched chicks were also analyzed.In this study, two doses (35 and 70 μmol/egg) of L-Leu were administered in ovo administration of L-Leu dose-dependently reduced RT and plasma thyroxine (T4) level just after hatching. In post-hatched neonatal broiler chicks, however, the higher dose of L-Leu administered in ovo significantly increased RT without affecting BW gain. In chicks that had been exposed to heat stress, the RT was significantly lowered by in ovo administration of L-Leu (high dose) in comparison with the control chicks under the same high ambient temperature .It was found that In ovo administration of L-Leu in a high dose contributed to an increased daily body temperature and afforded thermotolerance under HT in neonatal broiler chicks. Heat stress presents an increasing challenge for poultry production across the world since chickens are very sensitive to high ambient temperature (HT) . The HT In ovo administration of L-Leu (35 μmol/egg) on embryonic day (ED) 7 was then found to cause hypothermia at hatching, and also to afford thermotolerance in young broiler male chicks under HT [in ovo to develop thermoregulatory functions in broiler chicks is still unknown. The thyroid hormones thyroxine (T4) and triiodothyronine (T3) are associated with metabolic rate, which affects the regulation of body temperature [in ovo, as well as their effects on thyroid hormone levels at hatching in broiler chicks.Leucine (Leu) has been demonstrated to play critical roles in physiological regulation, including in protein synthesis, in reducing muscle damage, and in glucose or lipid metabolic activity ,11. In ounder HT . Howeverperature ,14. It wperature . The firin ovo compared with control chicks at 180 min of a heat challenge (35°C±1°C) [In ovo administration of a high dose of L-Leu could be expected to produce more potential effects of L-Leu in terms of affording thermotolerance in broilers and it could also help to clarify the mechanisms whereby L-Leu develops heat-resistant broilers. The second aim of this study was to investigate the effect of in ovo administration of a high dose (70 μmol/egg) of L-Leu on daily body temperature and BW, as well as on food intake, plasma metabolites and thermotolerance in chicks under heat stress.Plasma triacylglycerol, non-esterified fatty acid (NEFA) and ketone body levels were significantly increased, and in addition, food intake significantly decreased, in male chicks administered with L-Leu in ovo administration was the same as reported previously [g at 4°C for 4 min in order to separate out the plasma.In Experiment 1, forty-five fertilized broiler eggs were purchased from a local hatchery  and placed in an incubator . The eggs were divided into three groups (n = 15/group) based on the initial egg weight, with the resulting groups being as uniform possible. The groups were as follows: control (sterile water injection in the amount of 500 μL/egg); L-Leu low dose (35 μmol/500 μL/egg); and L-Leu high dose (70 μmol/500 μL/egg)  = 65.2 g; L-Leu (high) = 65.3 g). The eggs were incubated at a temperature of 37.6°C, at 58% to 68% relative humidity, and with auto-turning per h. The process of eviously . The chiin ovo administration processes were the same as described above in Experiment 1. After hatching, the chicks were housed in groups in metal cages  under a control thermoneutral temperature (CT) with the following temperature schedule: 30°C for 1 to 4 days, and 28°C for 5 to 10 days. The chicks were provided with free access to food  and water as well as continuous light. In previous studies [in ovo administration of L-Leu was found to afford thermotolerance in young male broiler chicks. At 2 days old, therefore, the males were separated, by means of sexing through feather identification, for further experimental procedures to be carried out on them. The 19 males  were subjected to a post-hatch experiment. At 8 days old, the males were individually isolated in small metal cages . Since in our previous study the body temperature in birds administered with L-Leu in ovo was found to have significantly decreased 120 min after starting the heat stress during 180 min of heat exposure [In Experiment 2, fifty-four eggs were divided into two groups based on their initial weight. The experimental groups were: control  and L-Leu high dose . The incubation and This study was performed in accordance with the guidelines for animal experiments in the Faculty of Agriculture of Kyushu University and complied with Law No. 105 and Notification No. 6 of the Japanese government.3 and T4 concentrations. Briefly, plasma samples were spiked with 13C6-T3  as an internal standard and were added to a mixture of 5% NH4OH and acetonitrile , and mixed thoroughly in a vortex. Samples were loaded onto SPE columns  and extracted by using 5% formic acid in methanol. After evaporation, the samples were reconstituted in 2 mM ammonium acetate/0.1% formic acid/50% methanol for LC-MS/MS analysis. The samples were analyzed on a QTRAP 5500 LC-MS/MS system  connected to a Shimadzu LC 20A HPLC system. The multiple reaction monitoring transitions in positive ionization mode were as follows: for T3, 651.8>605.8; for T4, 777.8>731.8; for 13C6-T3, 657.9>611.9.Plasma samples were subjected to LC-MS/MS analysis to determine T3), lactic acid (LA), lactate dehydrogenase (LDH), immunoglobulin A (IgA) and ketone bodies were examined using a reagent kit provided by the manufacturer to run in a Beckman Coulter AU480 automatic biochemistry analysis system .Plasma NEFA, ammonia . Significant differences were denoted as p<0.05. Data were expressed as mean±standard error of the mean. All data in each group were first subjected to a Thompson’s rejection test to eliminate outliers (p<0.01) [t-test.The data concerning RT and relative BW, as well as thyroid hormones at hatching, were statistically analyzed by one-way analysis of variance (ANOVA) following a post-hoc analysis using Holm-Sidak’s multiple comparisons test that was carried out when a significant difference was detected. The data concerning daily RT and BW, as well as RT changes under heat stress, were statistically analyzed by a two-way repeated-measures ANOVA following a post-hoc analysis using Holm-Sidak’s multiple comparisons test that was carried out when a significant interaction was detected, where the main effects were (p<0.01) , and theIn ovo administration of L-Leu dose-dependently decreased (p<0.05) RT at hatching in comparison with control-administered chicks. RT was found to have significantly decreased in the high-dose L-Leu group, and it showed a tendency to decrease (p = 0.078) in the low-dose L-Leu group, compared with the control group (in ovo administration of L-Leu did not affect the relative BW of chicks at hatching (3 was not affected by in ovo administration of L-Leu (in ovo administration of a high dose of L-Leu significantly (p<0.05) reduced the plasma T4 concentration and increased (p<0.05) the ratio of T3/T4 compared with the control group and the L-Leu (low-dose) group (ol group . Howeverhatching . The thyof L-Leu . Howevere) group .In ovo administration of L-Leu significantly increased RT, but not BW gain, compared with the control treatment within the experimental days  increased with age from 3 days old, but the values for RT were constant from 4 days old. tal days . HT caust stress , while funder HT . Plasma nt study .in ovo and examined their effects on RT, BW, and thyroid hormone levels at hatching in Experiment 1. The effects of high-dose administration of L-Leu (70 μmol/egg) on changes in daily RT and BW gain under CT, as well as on thermotolerance and food intake under HT, were examined in post-hatched chicks in Experiment 2. Accordingly, we confirmed that in ovo administration of a high dose of L-Leu has a stronger influence on regulation of body temperature at hatching. To the best of our knowledge, this is the first report to show that daily body temperature is higher in post-hatched chicks as a result of the in ovo administration of an amino acid—L-Leu. The plasma metabolites were also confirmed not to have contributed to the L-Leu-mediated improvement in thermotolerance.In the current study, we administered two doses (35 and 70 μmol/egg) of L-Leu in ovo administration of L-Leu (35 μmol/egg) once, on ED 7, has a strong influence in terms of stimulating embryonic metabolic activity and providing thermotolerance in post-hatched male broiler chicks under heat stress [In ovo administration of L-Leu dose-dependently decreased RT at hatching. The tendency that was observed (p = 0.078) for RT to decline in the low-dose group compared with the control group might have been due to the sex-specific effects of in ovo administration of L-Leu, since it has been reported that females are more responsive to L-Leu administration in terms of showing a reduced RT at hatching [in ovo administration of L-Leu on ED 7 stimulated metabolic activity and lipid metabolism during the middle stages of embryogenesis [in ovo administration of L-Leu, which resulted in a lower RT at hatching in birds administered with L-Leu in ovo.Recently, we reported that t stress . In the hatching , which rhatching . Previou p = 0.07 for RT t4 and T3) increase with the process of embryonic development and reach a peak before hatching [4 and T3 have been documented to stimulate the metabolic rate in birds, except at the neonatal stage, and to play a role in heat production as well as in adaptive variation in heat exposure [4 concentration decreased in association with lower body temperature in the high-dose group in comparison with control birds at hatching, and this finding was similar to that in a previous study, where the TM-mediated thyroid hormone and body temperature were found to have declined at hatching [3 and T4 did not influence body temperature, the inhibition of the hypothalamus-pituitary-adrenal axis was found to lower body temperature [4 is weaker than that of T3 [4 and T3) was not affected by in ovo administration of L-Leu under short-term heat stress, and that even thermotolerance was improved in 4-week-old male broilers (unpublished data). Together, these findings suggest that the reduced body temperature found in the high-dose group at hatching might not be explained by the lower level of plasma T4.Thyroid hormones  were not affected at 120 min of heat exposure (in ovo (unpublished data). Therefore, it could be speculated that in ovo administration of L-Leu might cause some epigenetic modulations in certain neural factors in broiler chicks to afford thermotolerance since the brain is the center of thermoregulation [Heat exposure significantly increased body temperature, which is a normal physiological response under heat stress . Increasexposure , which sf chicks ,8,27. Hif chicks . A previexposure , Table 1gulation . Future in ovo administration of L-Leu caused hypothermia and a reduced plasma T4 level at hatching, as well as enhancing the daily thermoregulation process in neonatal chicks and affording thermotolerance. The present study clarified that food-intake regulation and lipid metabolism did not contribute to the L-Leu-mediated improvement in thermotolerance under short-term heat stress. A future study will reveal the biochemical and molecular mechanisms whereby L-Leu improves thermotolerance.In conclusion, the stronger effects of high-dose


Lower photocurrent was observed on elevated ridges around defects (such as V-pits) in the films with Nanoscale structure has a large effect on the optoelectronic properties of InGaN, a material vital for energy saving technologies such as light emitting diodes. Photoconductive atomic force microscopy (PC-AFM) provides a new way to investigate this effect. In this study, PC-AFM was used to characterise four thick (∼130 nm) In With a direct bandgap which can be tuned from ultraviolet 3.5 eV) to infrared (0.7 eV)  with incs (LEDs) ,3, in wh.5 eV to Although the production of GaN single-crystal substrates is possible , the ext108 cm−2 , and theA recent cathodoluminescence study on thick InGaN layers has demonstrated that the core region of such threading dislocations is associated with enhanced light emission ; the autFor example, conductive-AFM (C-AFM) analysis of pure GaN has demonstrated that certain threading dislocations act as current leakage paths under reverse bias ,9,10,11,2 atmosphere [C-AFM studies are complicated by the formation of an insulating layer wherever current flow occurs on the sample surface ,15, withmosphere , and theA recent study by Kim et al.  used C-AThe recently developed technique of photoconductive AFM (PC-AFM) builds upon C-AFM by including a one-sun light source incident on the lower surface of a transparent sample, underneath the tip location. Here “one-sun” refers to the light approximating solar illumination, since PC-AFM is commonly used on materials for photovoltaic applications . This giIn this study, in addition to providing additional information on the properties of dislocations in thick InGaN layers, one aim was to gauge the usefulness of PC-AFM as an InGaN characterisation technique, highlighting not only its strengths but also some of its challenges and pitfalls. The samples studied were thick (∼130 nm) InGaN films with various indium contents grown on c-plane sapphire. Such thick InGaN layers have potential applications in solar cells   to determine their bulk indium content, thickness, and relaxation—results are given in 0.12Ga0.88N film , so no comparison can clearly be made between samples. Carrying out more repeats would be difficult due to the trade-off between identifying tip changes and further damaging the tip to do so. Interestingly, G was always lower when the light was on, which is unexpected. This could be explained by the insulating layer that builds up on the surface once current starts to flow; higher initial current may lead to more insulating layer being generated, which would reduce the measured conductance. Conversely, Measuring I-V curves also provides a way to quantify nanoscale features such as the defect ridges, since the location at which the I-V curve is taken can be specified. A key consideration is that the ridges are not much larger than the tip radius, so any drift in the sample after the initial topography map is taken could lead to the tip missing the ridge and mistakenly taking an I-V curve of background material. 0.12Ga0.88N layer. Measured turn-on voltage was much higher on the ridges; the increases were 0.09Ga0.91N and In0.15Ga0.85N layers, focussing on a single V-pit cross-section in each. Comparison between these two samples is particularly apt since In0.09Ga0.91N is representative of the samples with ridges around pits and photocurrent drops (low indium content samples), while In0.15Ga0.85N had no ridges or drops.0.09Ga0.91N film in g = 0002 and Looking at the annular dark field (ADF) scanning TEM (STEM) image of the In0.15Ga0.85N V-pit. Focussing on x in InThe STEM-EDS maps provide indium content as atomic percent rather than as A key observation in this study has been how surface current flow is altered by elevated ridges around defects in the 5–12% InN samples; illuminated PC-AFM maps show that much less current is measured on these ridges than on the rest of the surface . In addi0.09Ga0.91N film. Hence the low current flow and higher These data are consistent with the STEM-EDS results in 0.09Ga0.91N sample. From this point the V-pit growth occurs due to slower growth rate on the exposed V-pits within MOCVD-grown QW structures have been analysed extensively in previous literature ,25,26,27Applying this growth mechanism to our thick layers, lower indium incorporation on the facets of the V-pit as it grows could directly explain the shape of the indium-deficient region in ADF-STEM. Hence the boundary plane around the low indium area is determined by the relative growth rate in the V-pit compared to the rate on the adjacent (0001) plane. This hypothesis is reinforced by the fact that V-pit sidewall QWs in multiple QW structures also have lower indium content than (0001) QWs , providi0.15Ga0.85N film can also help explain the lack of ridges and photocurrent drops in the PC-AFM results from the same sample. V-pit morphology in this film is significantly different from the In0.09Ga0.91N film, with no low indium region and many dislocations branching around the pit. Similar dislocation branching has been observed before in MOCVD-grown high indium content thick InGaN films [0.09Ga0.91N and In0.15Ga0.85N are likely related to the lack of ridges in the In0.15Ga0.85N layer.TEM results on the InaN films ,30. In paN films  analysed1 radiation with a double-bounce Ge (220) asymmetric monochromator and a double-bounce analyser.All samples were grown on c-plane sapphire substrates by MOCVD in a Thomas Swan 6 × 2 in. close-coupled showerhead reactor; the exact method is described in more detail elsewhere . All areTEM samples were prepared using standard mechanical polishing followed by Arr method .In summary, the main observation was consistently lower current measured on ridges around defects in the 5–12% InN samples, with TEM suggesting this effect is related to sub-surface indium-deficient regions present around V-pits, which form due to lower indium incorporation on pit facets during growth.0.15Ga0.85N) was found to be significantly different from the others, with no ridges and lower measured current in the surface valleys. TEM analysis revealed that this sample’s V-pit morphology was also substantially different, with extra branching dislocations and no indium-deficient zone, which may explain why no ridges are present around the pits.The high indium content film (InWe can also comment on the applicability of PC-AFM to further characterise the InGaN materials system. Illumination allowed for current maps to be obtained more easily at a lower applied bias, since the turn-on voltage significantly decreased with light exposure. In general, PC-AFM suffers from issues such as tip inconsistency and induced sample surface changes, but its ability to observe new phenomena in a III-nitride semiconductor system has been demonstrated in this study.


Magnetic resonance imaging (MRI) has played important roles in neuro-image analysis while histological data remain crucial as a gold standard to guide and validate such analyses. With cellular-scale resolution, the BigBrain atlas offers 3D histology of a complete human brain, and is highly valuable to the research and clinical community. To bridge the insights at macro- and micro-levels, accurate mapping of BigBrain and established MRI brain atlases is necessary, but the existing registration is unsatisfactory. The described dataset includes co-registration of the BigBrain atlas to the MNI PD25 atlas and the ICBM152 2009b atlases (symmetric and asymmetric versions) in addition to manual segmentation of the basal ganglia, red nucleus, amygdala, and hippocampus for all mentioned atlases. The dataset intends to provide a bridge between insights from histological data and MRI studies in research and neurosurgical planning. The registered atlases, anatomical segmentations, and deformation matrices are available at:  Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.9914126 The development of magnetic resonance imaging (MRI) has allowed sophisticated computational algorithms5 to reveal structural and functional variations in living brains due to neurological developments and disorders. Often averaged from multiple subjects, the newer MRI brain atlases9 provide high-quality anatomical and physiological information, which can be mapped to an individual’s brain to facilitate further analyses. Despite the advancements to improve resolution, MRI signals remain at macroscopic resolutions. The BigBrain atlas10 is a 3D digitized model of a human brain at a near-cellular 20 micrometer resolution. It is a unique tool to help integrate cytoarchitectural knowledge with MRI insights to study brain functions and to define anatomical structures that can be difficult to image in MRI  for clinical practice and research. To bridge histological data with MRI, an accurate mapping between BigBrain and MRI brain atlases is necessary. Previously, a nonlinear registration between BigBrain and the ICBM152-2009b symmetric brain template8 was provided at bigbrain.loris.ca. This co-registration was achieved by deforming BigBrain with an inverted intensity profile to a population-averaged T1 map that is co-registered to the MNI space, with the SyN algorithm11 and cross-correlation similarity metric. However, this anatomical alignment, especially for subcortical structures, is not satisfactory, likely due to the discrepancy in tissue contrasts between BigBrain and the T1 map. As a result, more accurate alignment is greatly beneficial for various studies and surgical planning.Brain atlases are essential tools in neuroimage analysis and in neurosurgery, where they provide the reference to help navigate the anatomical and physiological features of the brain. While the foundational histology-derived atlases, such as Talairach8, from the Montreal Neurological Institute (MNI) is one of the most influential tools in neuroimage analysis. MRI brain scans of 152 young adults at 1.5T were recruited to build the multi-contrast atlas, which includes T1w, T2w, and PDw contrasts, as well as probabilistic tissue maps and brain structural labels. After the initial edition with affine registration, the 2009 edition using group-wise nonlinear registration provides unbiased representation of the brain anatomy with sharp details. For this edition, both symmetric and asymmetric atlases were offered at the resolutions of 0.5 × 0.5 × 0.5 mm3 (ICBM2009b) and 1 × 1 × 1 mm3 (ICBM2009c).The ICBM152 brain atlas dataset9 are created to ensure the quality of neuroimage analysis and surgical planning. Aiming to facilitate the research and surgical treatment of Parkinson’s disease (PD), the MNI PD25 population-averaged atlases12 were constructed from 3T MRI scans13 of 25 PD patients, and contain five different image contrasts, including T1w, T2*w, T1-T2* fusion, phase, and an R2* map. The special T1-T2* fusion atlas has the general T1w contrast for most of the brain while preserving the subcortical structures, such as the basal ganglia, red nucleus, and dentate nucleus as shown in typical T2*w contrast, which often suffers from susceptibility artefacts near the cortical surface. Furthermore, the dataset is co-registered with a digitized histological atlas with 123 structures14 and probabilistic tissue maps.As both natural ageing and neurological disorders can influence the anatomical features of the brain , population-specific atlases16, T1w-to-T1w registration can be sub-optimal for subcortical structures  that are nearly invisible in T1w MRI. We therefore employed a two-stage multi-contrast registration procedure with the PD25 space as the medium, as shown in Fig. 17 and to help validate the final registration outcomes. We expect the described dataset to greatly benefit the clinical and research community.In the dataset described here, we introduce an accurate nonlinear registration of BigBrain to the symmetric and asymmetric versions of ICBM2009b atlas and the MNI PD25 atlas. As suggested by earlier studies17, and thus reduces the potential loss that is propagated to atlas-to-subject mapping. To simplify the notations for all atlases involved in this article, we refer to the symmetric and asymmetric versions of ICBM152 2009b release as ICBMsym and ICBMasym, respectively, and use the name BigBrainSym to call the original co-registered BigBrain atlas to the ICBM152 space as provided in the BigBrain 2015 release. To aid the readers, the list of short names for different atlases is provided in Table 3 resolution for BigBrainSym, and at 0.5 × 0.5 × 0.5 mm3 for the MNI PD25 and the ICBM2009b symmetric and asymmetric atlases. These structures include the putamen, caudate nucleus, globus pallidus pars externa (GPe), globus pallidus pars interna (GPi), nucleus accumbens (NAc), amygdala, thalamus, red nucleus (RN), substantia nigra (SN), subthalamic nucleus (STN), and hippocampus. The segmentation was performed using ITK-SNAP (itksnap.org) with the left and right side labelled separately. While the RN and the basal ganglia structures were labelled by the author TA and revised by YX, who is experienced in brain anatomy, the rest were completed by YX. Here, the hippocampus segmentation follows the protocol employed by DeKraker et al.18, and the amygdala and NAc labels follow the protocol by Pauli et al.6. The full list of segmented structures and their associated label numbers are provided in Table BigBrainSym, PD25, and ICBMsym at 0.5 × 0.5 × 0.5 mm3 resolution. The segmentations were compared against those by YX using Dice coefficient, and the results are shown in Table 3 with nearest-neighbourhood interpolation for comparison.Manual segmentations were used to facilitate atlas-to-atlas registration and validate the registration results. This approach ensures the optimal structural overlap in multi-modal registrationHere, the Dice coefficient (κ) for assessing inter-rater variability is computed by:Isyn−T2w was constructed as:IT1w and IT1-T2* are the T1w and T1-T2* PD25 atlases, Imask is the brain mask, and Max{I} is the maximum value within the image I. The resulting synthetic T2w PD25 atlas is shown in Fig. 13, T2w MRI is still commonly used in the clinic for surgical planning. The synthetic T2w template can help directly bridge clinical T2w MRI scans with the PD25 templates.As the MNI PD25 dataset primarily leverages the T2*w contrast to visualize the subcortical structures , direct mapping between the PD25 T1-T2* atlas and T2w MRI scans can be challenging due to differences in image contrasts when conventional similarity metrics  are used during nonlinear registration. To facilitate the inter-contrast registration, a synthetic T2w PD25 atlas, BigBrainSym to initiate the atlas-to-atlas registration as it provided a good starting point. There are two main difficulties in mapping BigBrainSym to the ICBM152 atlases. First, the reconstructed histological volume has a unique and different appearance from the ICBM152 atlases, making accurate nonlinear registration with conventional image similarity metrics  challenging. Second, tissue tear and distortion from histology handling created unrealistic morphology  and artifacts  that can adversely influence the mapping. To mitigate these issues, we implemented a two-stage multi-contrast strategy to warp between BigBrainSym and the ICBM152 atlases, by using the MNI PD25 space as an intermediate template and adding anatomical segmentations as shape priors to further guide the registration. More specifically, BigBrainSym was first nonlinearly registered to the PD25 space, which was then deformed to the ICBMsym or ICBMasym atlas. Lastly, the deformation fields from the two stages were concatenated, and used to resample the BigBrainSym to the ICBM152 space. For both stages, we used antsRegistration from the Advanced Normalization Tools  to achieve the image registration, and all images were processed in MINC2 format.We employed BigBrainSym and the T1-T2* PD25 atlas, we used this pair of images to achieve the registration in the first stage. Inherited from the data in the native histological space, BigBrainSym contains a few problematic examples of anatomical morphology and artefacts. Besides those mentioned earlier, BigBrainSym also has an oversized pineal gland and tectum - likely from tissue stretching during histological processing. To cope with these, the pineal gland was removed from BigBrainSym for registration to avoid over-stretching of local deformation, which can adversely affect the overall registration, and the tectum was segmented in addition to the 11 subcortical structures in both atlases to constrain the registration. The multi-class segmentations were placed in one image for each atlas and blurred by a Gaussian kernel with a full-width-at-half-maximum (FWHM) of 0.5 mm. Finally, they were used jointly with the atlases during registration. Here, we used Mattes mutual information and cross-correlation for the atlas pair (weight = 1) and the segmentations (weight = 0.8), respectively.Taking advantage of the contrast similarity between As shown in Fig. 19, and pair-wise nonlinear registration should sufficiently account for the anatomical differences. Lastly, we hope the resulting dataset can benefit both healthy and pathological population, as well as neurosurgical applications, such as deep brain stimulation (DBS). The inclusion of PD25 atlas, therefore, will be very beneficial.In the two-stage registration strategy, PD25 was used as an intermediate volume due to a few considerations. First, compared with the previous approach that uses intensity inversion and a T1 map as the intermediate volume, the T1-T2* fusion contrast of PD25 has much closer resemblance to BigBrain, particularly for the subcortical structures. This can facilitate automatic registration. Second, BigBrain was derived from a 65-year-old healthy male, whose age is within the range of PD25 cohort (age = 58 ± 7 years). The difference in brain atrophy patterns in normal aging and Parkinson’s disease without cognitive impairment (target group for PD25) is relatively smallBigBrain-to-PD25, PD25-to-ICBM152 (symmetric and asymmetric versions), and finally BigBrain-to-ICBM152 (symmetric and asymmetric versions) registrations. Additionally, as a reference, we also calculated the two metrics between BigBrainSym and ICBMsym.The quality of atlas registration was assessed with two widely employed approaches: (1) anatomical landmark  registration errors and (2) atlas-based subcortical segmentation accuracy. While the first metric evaluates the matching of distinct anatomical features, the latter validates the correspondence of subcortical structures. Both metrics were computed for et al.20. For the framework, anatomical landmarks were selected by eight experienced raters for BigBrainSym, ICBMsym, and ICBMasym, and the final landmark coordinates at each location was obtained by averaging the results from all raters after filtering out outlier points. Following the same protocol, the final anatomical landmarks for the MNI PD25 atlas were produced by five experienced raters based on the T1w atlas. The full details of the landmark picking protocols and the associated software can be found in the original AFIDs article20. Since for BigBrainSym, excessive tissue tear and distortion exists, we excluded the pineal gland and culmen from the original AFIDs protocol20 for registration validation. The 30 anatomical landmarks employed for registration validation are listed in Table To assess the atlas alignment with landmark registration errors, we used the anatomical fiducials (AFIDs) framework introduced by Lau The Dice coefficient (κ) metric was used to evaluate the quality of volumetric overlap between the native manual segmentation and the corresponding labels warped from another atlas. For smaller structures, such as the midbrain nuclei, values greater than 0.7 are usually accepted as good segmentations while for larger structures, values above 0.8 are preferred.PD25, ICBMsym, and ICBMasym atlases at three different resolutions . The subcortical segmentations  was used for image format conversion. Lastly, the dataset provides the nonlinear transformations for BigBrainSym-to-PD25, PD25-to-ICBMsym, PD25-to-ICBMasym, BigBrainSym-to-ICBMsym, and BigBrainSym-to-ICBMasym registrations. All these transformations are provided in MINC transformation format. The full dataset can be accessible at the Open Science Framework (OSF)21 (10.17605/OSF.IO/XKQB3), as well as the main project page for the MNI PD25 atlases at nist.mni.mcgill.ca/?p=1209. In addition, all manually segmented label maps and brain atlases are also made available in BIDS format at OpenNeuro.org22 (https://openneuro.org/datasets/ds002016/versions/1.0.0).The complete dataset includes deformed atlases, subcortical segmentations, and inter-atlas spatial transformations. More specifically, we supply the BigBrain atlas deformed to the BigBrainSym-to-PD25, PD25-to-ICBMasym, PD25-to-ICBMsym, BigBrainSym-to-ICBMasym, and BigBrainSym-to-ICBMsym, respectively. For comparison, in Table BigBrainSym vs. ICBMsym). In general, the introduced two-stage registration strategy resulted in a slightly better mean registration error (1.77 ± 1.25 mm) than the previous registration for BigBrain vs. ICBMsym (1.83 ± 1.47 mm), but by performing a pair-wise Wilcoxon signed rank test, this difference is not significant (p = 0.959). As mentioned in the original AFIDs article20, ventricular features  generally have a bit higher landmark placement errors, and landmark placement is also more difficult for individual subjects than population-averaged atlases. This can be even more challenging for histological data, where unnatural deformation and unique individual anatomical features exist. As shown in Table Landmark registration errors were computed for all individual anatomical landmarks and for the five sets of atlas-to-atlas registrations involved in this dataset. The results are shown in Table 1.83 ± 1.7 mm, butBigBrainSym is better aligned with the ICBM152 space than PD25 for certain landmarks, likely due to better population representativeness with a larger cohort and the fact that the BigBrain landmarks were tagged within the BigBrainSym atlas20, potentially making the these landmark placement more biased towards the ICBM152 space.When looking closer at all registration results, the averaged errors from MRI-to-MRI registrations were lower than those from BigBrain-to-MRI registrations. This is expected, since individual anatomical variability is more pronounced than group-averaged anatomy and inter-modality registration is more challenging. Also, in terms of landmark registration errors, BigBrainSym-to-PD25, PD25-to-ICBMasym, PD25-to-ICBMsym, BigBrainSym-to-ICBMasym, and BigBrainSym-to-ICBMsym, respectively. In contrast to a mean κ value of 0.77 from the original warping of BigBrain to the symmetric ICBM152 space, the new strategy greatly improved the subcortical alignment for all structures of interest. By adding manual labels in multi-contrast registration, the alignment of subcortical anatomy was relatively consistent across different registrations. This helps ensure the quality of atlas-to-subject registration for future investigations by reducing the accuracy loss in multi-modal atlas-to-atlas warping, and the same approach17 was employed earlier for histology-to-MRI registration. Although the manual segmentations were also used for validation, the improved deformation is substantial in terms of Dice coefficient measurements for the subcortical structures, and it is also evident by visual inspection in Fig. Dice coefficients were calculated for 11 pairs of anatomical structures as listed in Table We provide the refined deformation matrices in MINC transformation format to comply with the existing releases of the BigBrain dataset. The linear and original nonlinear transformations between the BigBrain atlas in native histological space and the symmetric ICBM152 space are available at ftp://bigbrain.loris.ca/BigBrainRelease.2015/3D_Volumes/MNI-ICBM152_Space. The data described in this article are distributed under the CC BY 4.0 License upon the consent from the original data owners, the Montreal Neurological Institute  and the Forschungszentrum Jülich . However, this exception to the existing BigBrain dataset does not alter the general term of that license for the use of BigBrain itself, which is under the CC BY-NC-SA 4.0 License.